A pharmaceutical composition containing honokiol and its use in preventing and treating non-alcoholic fatty liver disease

Abstract

The present invention discloses the use of a compound of Formula I, or its salts, stereoisomers, tautomers, deuterated derivatives, solvates, metabolites, prodrugs, or mixtures thereof, in the preparation of a medicament for preventing and treating nonalcoholic fatty liver disease. The compound of the present invention can significantly inhibit free fatty acid-induced lipid deposition in AML12 cells, improve hepatocyte steatosis, significantly alleviate ballooning, and reduce inflammatory cell infiltration. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a pharmaceutical composition comprising honokiol and use of the pharmaceutical composition in preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely associated with insulin resistance and genetic susceptibility. It is characterized by abnormal lipid deposition in the liver. Excluding other secondary factors such as excessive alcohol consumption, medications, and genetic diseases that cause fatty liver, its spectrum includes nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), and cirrhosis. NAFLD has become the most common chronic liver disease worldwide, with a prevalence of approximately 25-30% in the general adult population. NASH is the key pathological stage in the progression of NAFL to fibrosis and cirrhosis and is one of the main causes of hepatocellular carcinoma. NASH-related cirrhosis has become the leading cause of liver transplantation in women and people over 65 years old. Lifestyle changes (weight loss, exercise, and a healthy diet) remain the cornerstone of NAFLD treatment. Guidelines from multiple countries recommend the glucagon-like peptide-1 analogs semaglutide and pioglitazone for patients with type 2 diabetes and liver biopsy-proven NASH, and vitamin E for those with liver biopsy-proven NASH. However, long-term, high-dose vitamin E use is associated with an increased all-cause mortality and an increased incidence of prostate cancer in men, so its safety remains to be evaluated. In March 2024, the US Food and Drug Administration approved the liver-targeted thyroid hormone receptor β-selective agonist resmetrom for the treatment of NASH with significant fibrosis, but its long-term safety remains to be determined. Furthermore, preliminary clinical trials of new drugs such as pan-PPAR agonists, fibroblast growth factor 21 analogs, and the GLP-1 receptor / glucose-dependent insulinotropic polypeptide and GLP-1 receptor dual agonist (telportide) have shown promising results, but further clinical data are needed. The previously highly anticipated farnesoid X receptor agonist obeticholic acid has not been approved for NASH treatment in the US and has been withdrawn from the market in Europe, further highlighting the continued need for effective drug development for NAFLD.

[0003] The pathological mechanisms of NAFLD are complex, and current drug designs targeting single targets are likely one of the reasons why most drugs that show promise in preclinical studies fail in clinical trials. Traditional Chinese medicine (TCM) has a proven clinical basis for treating NAFLD, and ongoing clinical studies have also demonstrated the potential efficacy of certain TCM formulas or Chinese patent medicines for NAFLD. This is likely due to the multi-component, multi-target nature of TCM. Furthermore, some TCM extracts or monomers, such as total paeony glucosides from white peony root, geniposide from gardenia jasminoides, salvianolic acid B from salvia miltiorrhiza, and berberine from coptis chinensis, have also demonstrated promising interventional effects on NAFLD, but these studies await confirmation in clinical trials.

[0004] There are no reports in the prior art that the compound of formula I claimed in the present invention, or its salts, stereoisomers, tautomers, deuterated forms, solvates, metabolites, prodrugs, or mixtures thereof, or a composition comprising the foregoing substances, can be used to prevent and / or treat liver diseases such as non-alcoholic fatty liver disease (non-alcoholic steatohepatitis). Summary of the Invention

[0005] Based on this, the present invention provides a use of a compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0006]

[0007] wherein R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C8 alkyl group, a C2 to C8 alkenyl group, a C2 to C8 alkynyl group, a C1 to C8 fluoroalkyl group, a C2 to C8 fluoroalkenyl group, a C2 to C8 fluoroalkynyl group, a C1 to C8 chloroalkyl group, a C2 to C8 chloroalkenyl group, a C2 to C8 chloroalkynyl group, a C1 to C8 bromoalkyl group, a C2 to C8 bromoalkenyl group, a C2 to C8 bromoalkynyl group, a C1 to C8 iodoalkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 ...iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 alkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 alkyl group, a 10 Aryl, substituted C6-C 10 aryl, 3-8 membered heteroaryl, substituted 3-8 membered heteroaryl, monosaccharide, disaccharide or polysaccharide.

[0008] Further, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C3 alkyl group, a C2 to C3 alkenyl group, a C2 to C3 alkynyl group, a C1 to C3 fluoroalkyl group, a C2 to C3 fluoroalkenyl group or a C2 to C3 fluoroalkynyl group.

[0009] Furthermore, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, hydroxyl, methyl, ethyl or propyl.

[0010] Furthermore, the compound of formula I is the honokiol shown in formula II,

[0011]

[0012] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, comprising a compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof and gentisic acid or its salt, wherein the mass ratio of the gentisic acid or its salt to the compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof is (45-800):about 1.

[0013] Furthermore, the mass ratio between the gentisic acid or a salt thereof and the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof is about 769.2:about 1, or about 200:about 1, or about 50:about 1.

[0014] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising a compound of formula I or a salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0015] Furthermore, the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels and inhibiting lipid deposition in AML12 cells.

[0016] Furthermore, the liver tissue pathological abnormality includes one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning and inflammatory cell infiltration.

[0017] Furthermore, the pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels.

[0018] Furthermore, the liver tissue pathological abnormality includes one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning and inflammatory cell infiltration.

[0019] Furthermore, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

[0020] Beneficial effects of the present invention:

[0021] The compound of the present invention can significantly inhibit free fatty acid-induced lipid deposition in AML12 cells, improve fatty degeneration of liver cells, significantly alleviate ballooning, and reduce inflammatory cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0023] Figure 1 Schematic diagram of the structural formulas of magnolol, honokiol and honokiol.

[0024] Figure 2 Schematic diagram of the effects of gentisic acid and honokiol on the relative viability of AML12 cells after 24 hours of incubation. A. Relative cell viability; B. Intracellular triglyceride content. G: gentisic acid, O: honokiol. *P < 0.05, **P < 0.01, ****P < 0.0001.

[0025] Figure 3 Schematic diagram of the effects of gentisic acid and honokiol on nonalcoholic fatty liver disease in mice induced by a CDAHFD. A, HE staining of liver tissue, B, TG staining of liver tissue, C, serum ALT staining. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Gl, low-dose gentisic acid, Gm, medium-dose gentisic acid, Gh, high-dose gentisic acid, Ol, low-dose honokiol, Om, medium-dose honokiol, Oh, high-dose honokiol.

[0026] Figure 4 Schematic diagram of the effects of the GO formula and different ratios of gentisic acid and honokiol (UA-UF) on CDAHFD-induced nonalcoholic fatty liver disease in mice. A, HE staining of liver tissue, B, TG staining of liver tissue, C, serum ALT staining. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0028] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0029] The present invention is intended to encompass all alternatives, modifications, and equivalents that may come within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0030] Unless otherwise indicated, the following definitions used herein shall apply.For purposes of the present invention, the chemical elements are as per the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994, the entire contents of which are incorporated herein by reference.

[0031] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0032] In the present invention, the term "comprising" is synonymous with "including." As used herein, the terms "comprises," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0033] The term "salt" refers to a salt formed by a compound of formula I with an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by a compound of formula I with an inorganic base; or a methylamine salt, ethylamine salt or ethanolamine salt formed by a compound of general formula I with an organic base.

[0034] The term "stereoisomers" refers to compounds that have identical chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0035] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that are interconvertible via a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0036] When tautomerism (e.g., keto-enol tautomerism) occurs in the compounds of the present invention or their prodrugs, protection is claimed for both their individual forms (e.g., keto or enol forms) and mixtures thereof in any ratio. The same applies to their stereoisomers, e.g., enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0037] If necessary, tautomers can be separated according to methods known in the art (e.g., liquid chromatography). The same applies to their enantiomers, for example, by separation using a chiral stationary phase. In addition, enantiomers can be separated by conversion into diastereomers, i.e., coupling with an enantiomerically pure auxiliary compound, followed by separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of the compounds of the invention can be obtained by stereoselective synthesis using optically pure starting materials.

[0038] The term "deuterated compound" used in the present invention refers to a deuterium-containing compound generated by replacing one or more hydrogen atoms in the compound of formula I with deuterium.

[0039] As used herein, the term "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or mixtures thereof. The term "hydrate" refers to an association formed when the solvent molecule is water. When the solvent is water, the term "hydrate" may be used. In one embodiment, one molecule of a compound of the present invention may be associated with one water molecule, such as a monohydrate; in another embodiment, one molecule of a compound of the present invention may be associated with more than one water molecule, such as a dihydrate; and in yet another embodiment, one molecule of a compound of the present invention may be associated with less than one water molecule, such as a hemihydrate. It should be noted that the hydrates of the present invention retain the biological effectiveness of the non-hydrated form of the compound.

[0040] As used herein, the term "metabolite" refers to a product resulting from the in vivo metabolism of a specific compound or salt thereof. Metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized using assays such as those described herein. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention encompasses metabolites of the compound, including metabolites produced by contacting a compound of the invention with a mammal for a sufficient period of time.

[0041] The term "prodrug" as used herein refers to a compound that is converted in vivo into a compound represented by Formula I. Such conversion is affected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissues. The prodrug compounds of the present invention may be esters. In the prior art, esters that can be used as prodrugs include phenyl esters, aliphatic (C 1-24 ) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound of the present invention containing a hydroxyl group can be acylated to produce a prodrug form of the compound. Other prodrug forms include phosphate esters, such as these phosphate ester compounds, which are obtained by phosphorylating a hydroxyl group on the parent compound.

[0042] Unless otherwise stated, all suitable salts, stereoisomers, tautomers, deuterated forms, solvates, metabolites and prodrugs of the compounds of the invention are included within the scope of the invention.

[0043] In the present invention, the term "C1 to C8" refers to a group having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. "3-8 membered" refers to a group having 3-8 ring atoms, and so on.

[0044] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety, for example, the term "C1 to C8 alkyl group" refers to a straight or branched alkyl group having 1 to 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0045] In the present invention, the term "alkenyl" refers to a straight chain or branched hydrocarbon moiety containing at least one double bond. For example, the term "C2 to C8 alkenyl" refers to a straight chain or branched alkenyl group containing one double bond having 2 to 8 carbon atoms, including but not limited to ethenyl, propenyl, n-butenyl, and isobutenyl.

[0046] In the present invention, the term "alkynyl" refers to a straight or branched alkynyl group containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl and the like.

[0047] In the present invention, the term "aryl" refers to a hydrocarbon moiety containing one or more aromatic rings. For example, the term "C6-C 10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl and naphthyl.

[0048] Unless otherwise specified, the alkyl, alkenyl, alkynyl, heteroaryl and aryl groups described herein are substituted and unsubstituted groups, and possible substituents include, but are not limited to, hydroxy, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C6 alkoxy, aryl, heteroaryl, heteroaryloxy ... C10 alkylamino, C1-C20 dialkylamino, arylamino, diarylamino, C1-C10 alkylsulfamoyl, arylsulfamoyl, C1-C10 alkylimino, C1-C10 alkylsulfoimino, arylsulfoimino, mercapto, C1-C10 alkylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidino, urea, cyano, acyl, thioacyl, acyloxy, carboxyl and carboxylate groups.

[0049] In the present invention, the substitution is mono- or poly-substituted, and the poly-substituted is di-, tri-, tetra-, or penta-substituted. The di-substituted refers to having two substituents, and so on.

[0050] As described in the background technology section, there are no reports in the prior art that the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug, or mixture thereof, or a composition comprising the same, as claimed in the present invention, can be used to prevent and / or treat liver diseases such as non-alcoholic fatty liver disease (non-alcoholic steatohepatitis). To address the above problems, the present invention provides a use of a compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug, or mixture thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0051]

[0052] wherein R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C8 alkyl group, a C2 to C8 alkenyl group, a C2 to C8 alkynyl group, a C1 to C8 fluoroalkyl group, a C2 to C8 fluoroalkenyl group, a C2 to C8 fluoroalkynyl group, a C1 to C8 chloroalkyl group, a C2 to C8 chloroalkenyl group, a C2 to C8 chloroalkynyl group, a C1 to C8 bromoalkyl group, a C2 to C8 bromoalkenyl group, a C2 to C8 bromoalkynyl group, a C1 to C8 iodoalkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 ...iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 alkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C8 alkyl group, a 10 Aryl, substituted C6-C 10 aryl, 3-8 membered heteroaryl, substituted 3-8 membered heteroaryl, monosaccharide, disaccharide or polysaccharide.

[0053] In a preferred embodiment, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C3 alkyl group, a C2 to C3 alkenyl group, a C2 to C3 alkynyl group, a C1 to C3 fluoroalkyl group, a C2 to C3 fluoroalkenyl group or a C2 to C3 fluoroalkynyl group.

[0054] In a preferred embodiment, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, hydroxyl, methyl, ethyl or propyl.

[0055] In a preferred embodiment, the compound of formula I is honokiol as shown in formula II,

[0056]

[0057] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, comprising a compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof and gentisic acid or its salt, wherein the mass ratio of the gentisic acid or its salt to the compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof is (45-800):about 1.

[0058] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0059] In the present invention, when mass ratio or other value or parameter is expressed in terms of a range, a preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "45 to 800" is disclosed, the described range should be interpreted as including the range of "45 to 800", "45 to 600", "45 to 400", "45 to 200", "45 to 100", "100 to 800", "100 to 600", "100 to 400", "100 to 200", "200 to 800", "200 to 600", "200 to 400", "400 to 800", "400 to 600", "600 to 800" and the like. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0060] In a preferred embodiment, the mass ratio of the gentisic acid or a salt thereof to the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof is about 769.2:about 1, or about 200:about 1, or about 50:about 1.

[0061] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 769.2" includes ±5% of 769.2, or from 730.74 to 807.66; "about 200" includes ±5% of 200, or from 190 to 210; "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0062] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising a compound of formula I or a salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0063] In a preferred embodiment, the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or mixture thereof prevents and treats non-alcoholic fatty liver disease by one or more of the following means: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels and inhibiting lipid deposition in AML12 cells.

[0064] In a preferred embodiment, the liver tissue pathological abnormality includes one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning, and inflammatory cell infiltration.

[0065] In a preferred embodiment, the pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels.

[0066] In a preferred embodiment, the liver tissue pathological abnormality includes one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning, and inflammatory cell infiltration.

[0067] In a preferred embodiment, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

[0068] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0070] The above-mentioned features of the present invention or the features described in the embodiments may be combined in any combination. All features disclosed in this patent specification may be used in any combination, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal, or similar purpose. Therefore, unless otherwise specified, the features disclosed are only general examples of equal or similar features.

[0071] Example 1 Effects of Honokiol, Gentisic Acid, Magnolol, and Honokiol on Free Fatty Acid-Induced Steatosis in AML12 Mouse Hepatocytes

[0072] 1. Experimental Materials

[0073] 1.1 Culture medium, reagents, and drugs

[0074] DMEM:F12 (Gibco, catalog number: 11320033), the basic culture medium contains 10% fetal bovine serum, 1% ITS Liquid Media Supplement (Sigma, I3146), 40ng / ml dexamethasone (Sigma, catalog number D4902-100mg), and 1% double antibody.

[0075] Oleic acid (Sigma, Catalog No. O1383) and palmitic acid (Sigma, Catalog No. A602448-0050) were prepared according to the reference method (Song Yuling, 2023) (Li Ran, 2022) and used as needed.

[0076] Honokiol (catalog number: AB3389, purity 98%), magnolol (catalog number: AB0682, purity 98%), honokiol (catalog number: AB1042, purity 98%), and gentisic acid (catalog number: AB1077, purity 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0077] The structural formulas of honokiol, magnolol and honokiol are as follows Figure 1 shown.

[0078] CCK8 kit (Biyuntian, cat. no. C0038) and cell triglyceride detection kit (Beijing Pulilai, cat. no. E1013) were used according to the instructions.

[0079] 1.2 Cell lines

[0080] AML12 mouse hepatocyte cell line was purchased from Shanghai Meiyan Biotechnology Co., Ltd.

[0081] 2. Modeling and grouping

[0082] 2.1 Cytotoxicity test:

[0083] The cells were seeded in 96-well plates, and the culture medium was replaced when the cells grew to more than 80%.

[0084] 2.1.1 Cytotoxicity test of magnolol and gentisic acid

[0085] AML12 cells were divided into blank control group, honokiol groups (200, 100, 50, 25 μM), and gentisic acid groups (200, 100, 50, 25 μM), with 10 wells in each group. The corresponding drugs were added and incubated for 24 hours. The cell viability was detected by CCK8 kit (Biyuntian, Cat. No.: C0038).

[0086] 2.1.2 Cytotoxicity experiments of magnolol, magnolol, and magnolol

[0087] AML12 cells were divided into blank control group, honokiol group (50 μM), magnolol group (50 μM), and honokiol group (50 μM), with 10 wells in each group. The corresponding drugs were added and incubated for 24 h, and the cell viability was detected by CCK8 kit.

[0088] 2.2. Fat deposition experiment in AML12 cells

[0089] AML-12 cells were cultured in 6-well plates and divided into normal, model, gentisic acid (10 μM), honokiol (10 μM), honokiol (10 μM), and magnolol (10 μM) groups, with 6 wells per group. Except for cells in the normal group, cells in all other groups were stimulated with palmitic acid (0.6 mM) and oleic acid (0.17 mM) for 24 hours (Zhou, Pang, Tripathi, Ho, Widjaja, Shekeran, Cook, Suzuki, Diehl, Petretto, Singh, and Yen, 2022). Drug groups were also treated with the corresponding drugs for 24 hours. After 24 hours, the culture medium was aspirated, cells were washed twice with PBS, and 1 ml of 0.25% trypsin was added to digest the cells for 1 minute. Cells from each well were collected into a 15 mL EP tube and the volume was adjusted to 3 ml. Cell counts were performed, and the total cell number was calculated. Centrifuge at 3000 rpm for 5 minutes, discard the supernatant, and rinse the pellet twice with 1 ml of PBS. Resuspend the pellet in 1 ml of PBS and transfer it to a 1.5 ml EP tube. Centrifuge again to collect the pellet. Detect the intracellular TG content according to the instructions of the Cell Triglyceride Detection Kit.

[0090] 3. Experimental results

[0091] 3.1 Cytotoxicity of Gentisic Acid, Honokiol, Honokiol, and Magnolol

[0092] Gentisic acid showed no cytotoxicity, honokiol showed cytotoxicity at a concentration of 100 μM, honokiol and magnolol showed cytotoxicity at a concentration of 50 μM, and honokiol, magnolol, and honokiol showed no cytotoxicity at a concentration of 10 μM (e.g. Figure 2 (as shown in A).

[0093] 3.2 Effects of gentisic acid, honokiol, magnolol, and honokiol on free fatty acid-induced lipid deposition in AML12 cells

[0094] Gentisic acid and honokiol at a concentration of 10 μM significantly inhibited free fatty acid-induced lipid deposition in AML12 cells (such as Figure 2 B). Magnolol and honokiol showed cytotoxicity at a concentration of 50 μM (as shown in Figure 2 A), but no inhibitory effect was observed on lipid deposition in AML12 cells induced by free fatty acids at a concentration of 10 μM (as shown in Figure 2 B).

[0095] Example 2 Effects of Magnolia officinalis phenol and gentisic acid on mice with non-alcoholic fatty liver disease

[0096] 1. Experimental Materials

[0097] 1.1 Experimental Animals

[0098] Male SPF C57BL / 6 mice, 6-8 weeks old, were housed in a 22-25°C environment with a relative humidity of 55-60% under natural light and with free access to drinking water.

[0099] 1.2. Medication

[0100] Gentisic acid and magnolol.

[0101] 1.3 Reagents

[0102] Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD, Catalog No.: A06071302, Research Diets Inc), triglyceride kit (Catalog No.: A010017, Zhejiang Dongou Diagnostic Products Co., Ltd.).

[0103] 2. Experimental methods

[0104] 2.1 Animal Model Preparation and Drug Administration

[0105] After one week of adaptive feeding, the mice were randomly divided into a normal control group, a model group, a high-dose (200 mg / kg body weight), a medium-dose (100 mg / kg body weight), and a low-dose (50 mg / kg) gentisic acid group, a high-dose (2 mg / kg), a medium-dose (1 mg / kg body weight), and a low-dose (0.5 mg / kg body weight) honokiol group, and an obeticholic acid control group, with eight mice in each group. The normal control group was fed a standard diet for 6 weeks, while the remaining groups were fed a CDAHFD diet for 6 weeks. Starting from the third week, the high-, medium-, and low-dose gentisic acid groups, the high-, medium-, and low-dose honokiol groups, and the obeticholic acid control group were given the corresponding drugs. The normal control group and the model group were gavaged with an equal amount of sterile drinking water. Mice were collected at the end of the sixth week.

[0106] 2.2 Sample Collection

[0107] 2.2.1 Serum

[0108] At the end of the sixth week, the mice were fasted for 12 hours, their eyeballs were removed to collect blood, and then they were killed by cervical dislocation. The blood was kept at 4°C for 4 hours, centrifuged at 3000 rpm for 15 minutes, and 200 μL of serum was collected in a 1.5 ml eppendorf tube and stored at -80°C.

[0109] 2.2.2 Liver

[0110] The mouse liver was excised from the abdominal cavity, rinsed with saline, and then dried on filter paper before being photographed and weighed. Two sections of liver tissue, approximately 1 cm x 1 cm x 1 cm, were removed from the central lobe of the liver. One section was placed in a pathology box and stored in formalin. The remaining liver tissue was quickly frozen in liquid nitrogen and stored at -80°C.

[0111] 2.3、Indicator detection

[0112] 2.3.1. Hematoxylin and eosin (H&E) staining of liver tissue

[0113] Liver tissue paraffin sections (4 μm) were stained using a hematoxylin-eosin staining kit (Nanjing Jiancheng Bioengineering Institute, catalog number C0105). Liver tissue paraffin sections (4 μm) were baked at 70°C for 40 minutes in a slide oven (LEICA, Germany, model HI1220) and dewaxed in the following order: xylene, 10 minutes; xylene, 10 minutes; anhydrous ethanol, 5 minutes; anhydrous ethanol, 2 minutes; 95% ethanol, 2 minutes; 85% ethanol, 2 minutes; 70% ethanol, 2 minutes; tap water, three times; double-distilled water, three times. The sections were then stained in the following order: hematoxylin stain, 15 minutes; tap water, three times; double-distilled water, three times; hydrochloric acid alcohol, 3 seconds; tap water, three times; double-distilled water, three times; eosin, 10 seconds; 95% ethanol, 1 minute; 95% ethanol, 1 minute; anhydrous ethanol, 1 minute; anhydrous ethanol, 1 minute; xylene, 1 minute; xylene, 1 minute. Neutral resin mounting medium (Sinopharm Chemical Reagent Co., Ltd., catalog number: E675007-00100) was used for mounting. All pathological slides were scanned and archived using a digital radiograph (LEICA, Germany, model: SCN400).

[0114] 2.3.2. Liver triglyceride (TG) detection

[0115] Before measuring liver TG, prepare liver tissue homogenate in advance: weigh 50 mg of liver tissue and place it in a 2 mL centrifuge tube containing 3 steel balls, add 375 μL of anhydrous ethanol and 375 μL of acetone, and grind it with an automatic sample grinder at 65 Hz for 60 s*3 times to obtain liver tissue homogenate. Let it stand in a 4°C refrigerator overnight, and centrifuge it the next day for 15 minutes (4°C, 3000 rpm / min). Take the supernatant for the determination of TG content according to the instructions.

[0116] 2.3.3 Serum ALT detection

[0117] Serum ALT was detected using a kit according to the instructions.

[0118] 3. Experimental results

[0119] 3.1. Pathological changes of mouse liver tissue

[0120] After H&E staining of liver tissue, it was observed under the microscope that the hepatocytes of mice in the normal control group had normal morphology, clear hepatic lobule structure, and neatly arranged hepatic cords. The hepatocytes of mice in the model group showed varying degrees of swelling, with huge fatty vacuoles in the cytoplasm, the hepatocyte nuclei squeezed to the edge, accompanied by ballooning of hepatocytes, and inflammatory cell infiltration in some portal areas and lobules. The fatty degeneration and ballooning of hepatocytes in mice in the medium and high dose groups of gentisic acid, and the medium and high dose groups of magnolia officinalis phenol were significantly alleviated, and the infiltration of inflammatory cells was reduced. The fatty degeneration and ballooning of hepatocytes in mice in the obeticholic acid control group were also significantly alleviated, and inflammatory cells were rare (such as Figure 3 (as shown in A).

[0121] 3.2 TG Content in Mouse Liver

[0122] The results of liver tissue TG content determination also showed that the TG content of liver tissue of mice in the model group was significantly higher than that in the normal control group, while the TG content of liver tissue of mice in the medium and high dose gentisic acid groups, the medium and high dose honokiol groups and the obeticholic acid group was significantly lower than that in the model group. There was no significant difference among the drug groups (such as Figure 3 B).

[0123] 3.3 Mouse serum ALT

[0124] Compared with the normal group, the serum ALT activity of the model group mice was significantly increased, and the serum ALT activity of the mice in the medium and high dose gentisic acid groups, the low, medium and high dose honokiol groups and the obeticholic acid intervention group was significantly lower than that of the model group. There was no significant difference between the drug groups (such as Figure 3 C).

[0125] Example 3 Pharmacodynamic screening experiment of the combination of magnolia bark phenol and gentisic acid

[0126] 1. Experimental Materials

[0127] 1.1 Experimental Animals

[0128] Male SPF C57BL / 6 mice, 6-8 weeks old, were housed in a 22-25°C environment with a relative humidity of 55-60% under natural light and with free access to drinking water.

[0129] 1.2. Medication

[0130] Gentisic acid and magnolol.

[0131] 1.3 Reagents

[0132] Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD, Catalog No.: A06071302, Research Diets Inc), triglyceride kit (Catalog No.: A010017, Zhejiang Dongou Diagnostic Products Co., Ltd.).

[0133] 2. Experimental methods

[0134] 2.1 Experimental Design Method

[0135] Different proportions of gentisic acid and magnolia officinalis phenol were calculated according to the uniform design table U6*(6 4) table (as shown in Table 1), the two monomer components: gentisic acid (represented by "G") and magnolol (represented by "O") were used as the investigation factors, and each factor took 6 dose levels, with the upper limit of the G dose being 200 mg / kg and the upper limit of the O dose being 2 mg / kg, decreasing at a rate of 0.5, according to U6*(6 4 ) table uses the table (as shown in Table 2) to select the first and third columns, as shown in Table 3.

[0136] Table 1 Uniform design table U6*(6 4 )surface

[0137] Group 1 2 3 4 1 1 2 3 6 2 2 4 6 5 3 3 6 2 4 4 4 1 5 3 5 5 3 1 2 6 6 5 4 1

[0138] Table 2U6*(6 4 ) Table usage table

[0139] s Column number D 2 1,3 0.1875 3 1,2,3 0.2656 4 1,2,3,4 0.299

[0140] Table 3 Uniform design experimental plan

[0141] Group G (mg / kg) O (mg / kg) UA 6.25 0.25 UB 12.5 2 UC 25 0.125 UD 50 1 UE 100 0.5 UF 200 0.0625

[0142] In addition, the GO prescription group (gentisic acid = 100 mg / kg, honokiol = 0.13 mg / kg) was added on this basis.

[0143] 2.2 Animal Model Preparation and Drug Administration

[0144] After one week of adaptive feeding, the mice were randomly divided into a normal control group, a model group, a GO formula group, a GO formula with different ratios (i.e., a uniformly designed UA-UF group), and an obeticholic acid control group, with eight mice in each group. The normal control group was fed a standard diet for 6 weeks, while the remaining groups were fed a CDAHFD diet for 6 weeks. Starting from the third week, the GO formula group, the UA-UF group, and the obeticholic acid control group were given the corresponding medications. The normal control group and the model group were gavaged with an equal volume of sterile drinking water. Samples were collected at the end of the sixth week.

[0145] 2.3 Sample Collection

[0146] 2.3.1 Serum

[0147] At the end of the sixth week, the mice were fasted for 12 hours, their eyeballs were removed to collect blood, and then they were killed by cervical dislocation. The blood was kept at 4°C for 4 hours, centrifuged at 3000 rpm for 15 minutes, and 200 μL of serum was collected in a 1.5 ml eppendorf tube and stored at -80°C.

[0148] 2.3.2 Liver

[0149] The mouse liver was excised from the abdominal cavity, rinsed with saline, and then dried on filter paper before being photographed and weighed. Two sections of liver tissue, approximately 1 cm x 1 cm x 1 cm, were removed from the central lobe of the liver. One section was placed in a pathology box and stored in formalin. The remaining liver tissue was quickly frozen in liquid nitrogen and stored at -80°C.

[0150] 2.4、Indicator detection

[0151] 2.4.1. Hematoxylin and eosin (H&E) staining of liver tissue was the same as in Example 2.

[0152] 2.4.2. Liver triglyceride (TG) detection

[0153] Same as Example 2.

[0154] 2.4.3 Serum ALT detection

[0155] Same as Example 2.

[0156] 3. Experimental results

[0157] 3.1. Pathological changes of mouse liver tissue

[0158] After H&E staining of liver tissue, it was observed under the microscope that the hepatocytes of mice in the normal control group had normal morphology, clear hepatic lobule structure, and neatly arranged hepatic cords. The hepatocytes of mice in the model group showed varying degrees of swelling, with huge fatty vacuoles in the cytoplasm, the hepatocyte nuclei squeezed to the edge, accompanied by ballooning of hepatocytes, and inflammatory cell infiltration in some portal areas and lobules. The fatty degeneration and ballooning of hepatocytes in mice in the GO prescription group (gentisic acid = 100 mg / kg, magnolia officinalis phenol = 0.13 mg / kg), UC, UD, UE, and UF groups were significantly alleviated, and the infiltration of inflammatory cells was reduced. The fatty degeneration and ballooning of hepatocytes in mice in the obeticholic acid control group were also significantly alleviated, and inflammatory cells were rare (such as Figure 4 (as shown in A).

[0159] 3.2 TG Content in Mouse Liver

[0160] The results of liver tissue TG content determination also showed that the liver tissue TG content of mice in the model group was significantly higher than that in the normal control group, while the liver tissue TG content of mice in the GO prescription group, UC group, and UD group and obeticholic acid group was significantly lower than that in the model group. There was no significant difference between the drug groups (such as Figure 4 B).

[0161] 3.3 Mouse serum ALT

[0162] Compared with the normal group, the serum ALT activity of the model group mice was significantly increased, and the serum ALT activity of the GO prescription group, UC, UD, UE, UF group and obeticholic acid intervention group mice was significantly decreased compared with the model group. There was no significant difference among the drug groups (such as Figure 4 C).

[0163] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. Use of magnolol or its salt in the preparation of medicines for preventing and treating non-alcoholic fatty liver disease.

2. A pharmaceutical composition of honokiol or its salt and gentisic acid or its salt for preventing and treating non-alcoholic fatty liver disease, characterized in that: The mass ratio between the gentisic acid or its salt and the honokiol or its salt is (45-800): (0.95-1.05).

3. The pharmaceutical composition according to claim 2, characterized in that The mass ratio of the gentisic acid or its salt to the honokiol or its salt is (730.74-807.66):(0.95-1.05), or (190-210):(0.95-1.05), or (47.5-52.5):(0.95-1.05).

4. The pharmaceutical composition according to claim 2 or 3, characterized in that The non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

5. Use of a pharmaceutical composition comprising honokiol or a salt thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

6. The use according to claim 1, characterized in that The effects of the honokiol or its salt on preventing and treating non-alcoholic fatty liver disease are achieved through one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels, and inhibiting lipid deposition in AML12 cells.

7. The use according to claim 6, characterized in that The liver tissue pathological abnormalities include one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning and inflammatory cell infiltration.

8. The use according to claim 5, characterized in that The pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels.

9. The use according to claim 8, characterized in that The liver tissue pathological abnormalities include one or more of the following: hepatocyte fatty degeneration, hepatocyte ballooning and inflammatory cell infiltration.

10. The use according to any one of claims 1, 5 to 9, characterized in that The non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

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